The investigation of the major determinants of monoamine synthesis and turnover in vivo is of intense scientific interest because monoamine levels in the central nervous system (CNS) play critical roles in neuropsychiatric, neuroendocrine and cardiovascular diseases. Tyrosine hydroxylase, which requires tetra-hydrobioterin (BH4) as a cofactor, is known to be the rate-limiting enzymatic step in the synthesis of dopamine and norepinephrine. Current evidence suggests that in vivo rate of synthesis depends primarily on dopamine concentration (due to end-product inhibition) and hydroxylase cofactor (BH4) levels. Thus, cofactor levels in cerebrospinal fluid (CSF) are of interest. This study was undertaken to detect and monitor CSF hydroxylase cofactor activity using a phenylalanine hydroxylase assay. Our studies indicate hydroxylase cofactor activity declines with age in both normal and diseased patients. No significant alteration was found in CSF cofactor levels of schizophrenic or affective disorder patients. We have reported that patients with certain neurological diseases, most notably Parkinson's disease, have reduced levels of cofactor in their CSF.